Cargando…

TDP-43-Mediated Toxicity in HEK293T Cells: A Fast and Reproducible Protocol To Be Employed in the Search of New Therapeutic Options against Amyotrophic Lateral Sclerosis

Cytoplasmic TDP-43 aggregates are a hallmark of amyotrophic lateral sclerosis (ALS). Today, only two drugs are available for ALS treatment, and their modest effect prompts researchers to search for new therapeutic options. TDP-43 represents one of the most promising targets for therapeutic intervent...

Descripción completa

Detalles Bibliográficos
Autores principales: Lanznaster, Débora, Bourgeais, Jérôme, Bruno, Clement, Hergesheimer, Rudolf C., Thepault, Rose-Anne, Vourc’h, Patrick, Corcia, Philippe, Andres, Christian R., Herault, Olivier, Blasco, Hélène
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7016571/
https://www.ncbi.nlm.nih.gov/pubmed/31888078
http://dx.doi.org/10.3390/cells9010068
_version_ 1783497005522223104
author Lanznaster, Débora
Bourgeais, Jérôme
Bruno, Clement
Hergesheimer, Rudolf C.
Thepault, Rose-Anne
Vourc’h, Patrick
Corcia, Philippe
Andres, Christian R.
Herault, Olivier
Blasco, Hélène
author_facet Lanznaster, Débora
Bourgeais, Jérôme
Bruno, Clement
Hergesheimer, Rudolf C.
Thepault, Rose-Anne
Vourc’h, Patrick
Corcia, Philippe
Andres, Christian R.
Herault, Olivier
Blasco, Hélène
author_sort Lanznaster, Débora
collection PubMed
description Cytoplasmic TDP-43 aggregates are a hallmark of amyotrophic lateral sclerosis (ALS). Today, only two drugs are available for ALS treatment, and their modest effect prompts researchers to search for new therapeutic options. TDP-43 represents one of the most promising targets for therapeutic intervention, but reliable and reproducible in vitro protocols for TDP-43-mediated toxicity are lacking. Here, we used HEK293T cells transfected with increasing concentrations of TDP-43-expressing plasmid to evaluate different parameters of toxicity and alterations in cellular metabolism. Overexpression of TDP-43 induced aggregates occurrence followed by the detection of 25- and 35-kDa forms of TDP-43. TDP-43 overexpression decreased cell viability and increased cells arrested at G2/M phase and nuclear fragmentation. Analysis of the energetic metabolism showed a tendency to decrease oxidative phosphorylation and increase glycolysis, but no statistical differences were observed. Metabolomics revealed alterations in different metabolites (mainly sphingolipids and glycerophospholipids) in cells overexpressing TDP-43. Our data reveal the main role of TDP-43 aggregation in cellular death and highlight novel insight into the mechanism of cellular toxicity induced by TDP-43. Here, we provide a simple, sensitive, and reliable protocol in a human-derived cell line to be used in high-throughput screenings of potential therapeutic molecules for ALS treatment.
format Online
Article
Text
id pubmed-7016571
institution National Center for Biotechnology Information
language English
publishDate 2019
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-70165712020-03-04 TDP-43-Mediated Toxicity in HEK293T Cells: A Fast and Reproducible Protocol To Be Employed in the Search of New Therapeutic Options against Amyotrophic Lateral Sclerosis Lanznaster, Débora Bourgeais, Jérôme Bruno, Clement Hergesheimer, Rudolf C. Thepault, Rose-Anne Vourc’h, Patrick Corcia, Philippe Andres, Christian R. Herault, Olivier Blasco, Hélène Cells Article Cytoplasmic TDP-43 aggregates are a hallmark of amyotrophic lateral sclerosis (ALS). Today, only two drugs are available for ALS treatment, and their modest effect prompts researchers to search for new therapeutic options. TDP-43 represents one of the most promising targets for therapeutic intervention, but reliable and reproducible in vitro protocols for TDP-43-mediated toxicity are lacking. Here, we used HEK293T cells transfected with increasing concentrations of TDP-43-expressing plasmid to evaluate different parameters of toxicity and alterations in cellular metabolism. Overexpression of TDP-43 induced aggregates occurrence followed by the detection of 25- and 35-kDa forms of TDP-43. TDP-43 overexpression decreased cell viability and increased cells arrested at G2/M phase and nuclear fragmentation. Analysis of the energetic metabolism showed a tendency to decrease oxidative phosphorylation and increase glycolysis, but no statistical differences were observed. Metabolomics revealed alterations in different metabolites (mainly sphingolipids and glycerophospholipids) in cells overexpressing TDP-43. Our data reveal the main role of TDP-43 aggregation in cellular death and highlight novel insight into the mechanism of cellular toxicity induced by TDP-43. Here, we provide a simple, sensitive, and reliable protocol in a human-derived cell line to be used in high-throughput screenings of potential therapeutic molecules for ALS treatment. MDPI 2019-12-26 /pmc/articles/PMC7016571/ /pubmed/31888078 http://dx.doi.org/10.3390/cells9010068 Text en © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Lanznaster, Débora
Bourgeais, Jérôme
Bruno, Clement
Hergesheimer, Rudolf C.
Thepault, Rose-Anne
Vourc’h, Patrick
Corcia, Philippe
Andres, Christian R.
Herault, Olivier
Blasco, Hélène
TDP-43-Mediated Toxicity in HEK293T Cells: A Fast and Reproducible Protocol To Be Employed in the Search of New Therapeutic Options against Amyotrophic Lateral Sclerosis
title TDP-43-Mediated Toxicity in HEK293T Cells: A Fast and Reproducible Protocol To Be Employed in the Search of New Therapeutic Options against Amyotrophic Lateral Sclerosis
title_full TDP-43-Mediated Toxicity in HEK293T Cells: A Fast and Reproducible Protocol To Be Employed in the Search of New Therapeutic Options against Amyotrophic Lateral Sclerosis
title_fullStr TDP-43-Mediated Toxicity in HEK293T Cells: A Fast and Reproducible Protocol To Be Employed in the Search of New Therapeutic Options against Amyotrophic Lateral Sclerosis
title_full_unstemmed TDP-43-Mediated Toxicity in HEK293T Cells: A Fast and Reproducible Protocol To Be Employed in the Search of New Therapeutic Options against Amyotrophic Lateral Sclerosis
title_short TDP-43-Mediated Toxicity in HEK293T Cells: A Fast and Reproducible Protocol To Be Employed in the Search of New Therapeutic Options against Amyotrophic Lateral Sclerosis
title_sort tdp-43-mediated toxicity in hek293t cells: a fast and reproducible protocol to be employed in the search of new therapeutic options against amyotrophic lateral sclerosis
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7016571/
https://www.ncbi.nlm.nih.gov/pubmed/31888078
http://dx.doi.org/10.3390/cells9010068
work_keys_str_mv AT lanznasterdebora tdp43mediatedtoxicityinhek293tcellsafastandreproducibleprotocoltobeemployedinthesearchofnewtherapeuticoptionsagainstamyotrophiclateralsclerosis
AT bourgeaisjerome tdp43mediatedtoxicityinhek293tcellsafastandreproducibleprotocoltobeemployedinthesearchofnewtherapeuticoptionsagainstamyotrophiclateralsclerosis
AT brunoclement tdp43mediatedtoxicityinhek293tcellsafastandreproducibleprotocoltobeemployedinthesearchofnewtherapeuticoptionsagainstamyotrophiclateralsclerosis
AT hergesheimerrudolfc tdp43mediatedtoxicityinhek293tcellsafastandreproducibleprotocoltobeemployedinthesearchofnewtherapeuticoptionsagainstamyotrophiclateralsclerosis
AT thepaultroseanne tdp43mediatedtoxicityinhek293tcellsafastandreproducibleprotocoltobeemployedinthesearchofnewtherapeuticoptionsagainstamyotrophiclateralsclerosis
AT vourchpatrick tdp43mediatedtoxicityinhek293tcellsafastandreproducibleprotocoltobeemployedinthesearchofnewtherapeuticoptionsagainstamyotrophiclateralsclerosis
AT corciaphilippe tdp43mediatedtoxicityinhek293tcellsafastandreproducibleprotocoltobeemployedinthesearchofnewtherapeuticoptionsagainstamyotrophiclateralsclerosis
AT andreschristianr tdp43mediatedtoxicityinhek293tcellsafastandreproducibleprotocoltobeemployedinthesearchofnewtherapeuticoptionsagainstamyotrophiclateralsclerosis
AT heraultolivier tdp43mediatedtoxicityinhek293tcellsafastandreproducibleprotocoltobeemployedinthesearchofnewtherapeuticoptionsagainstamyotrophiclateralsclerosis
AT blascohelene tdp43mediatedtoxicityinhek293tcellsafastandreproducibleprotocoltobeemployedinthesearchofnewtherapeuticoptionsagainstamyotrophiclateralsclerosis